The GH cells are rat pituitary derived cell lines (GH1, GC, GH3, and GH4) which produce growth hormone and or prolactin in response to many hormones. Preliminary data demonstrate that insulin and triiodothyronine (T3) under serum free conditions induce prolactin synthesis and prolactin mRNA levels 3- to 14-fold in GH3 cells. Together these hormones induce a dramatic 20- to 100-fold synergistic response. First, this research will attempt to define this response. The kinetics will be determined by in vitro transcription and in vivo [3H]uridine labeling studies using filter hybridization to determine the levels of total prolactin specific sequences. The specificity of the response for insulin or other insulin-like growth factors (IGF) will then be determined by dose response studies with insulin, insulin analogs, IGF-I and IGF-II. Studies to determine the effects of insulin and T3 on prolactin RNA processing and half-life are also proposed. Finally, the deinduction of the response after hormone removal will be investigated. Second, we will attempt to define possible insulin regulation of prolactin mRNA accumulation by studying effects of insulin and T3 on cis-active DNA regulatory sequences, trans-active regulatory molecules, or chromatin structure. Cis-active regulatory sequences will be studied by construction of a chimeric plasmid of 5' prolactin genomic DNA and the bacterial xanthine-guanine phosphoribosyl transferase (XGPRT) gene and studying hormonal regulation of XGPRT in cells transfected with this plasmid and with deletion mutants of this plasmid. Attempts will be made to identify any role of trans-acting regulatory molecules on this system by inhibitor studies and by two dimensional gel electrophoresis of labeled nuclear proteins. Finally, we will attempt to determine hormonal effects on the chromatin structure of the prolactin gene by DNase I sensitivity studies and examination of the methylatin state of the gene.